Deficiency in coatomer complex I causes aberrant activation of STING signalling

Coatomer complex I (COPI) mediates retrograde vesicular trafficking from Golgi to the endoplasmic reticulum (ER) and within Golgi compartments. Deficiency in subunit alpha causes COPA syndrome and is associated with type I IFN signalling, although the upstream innate immune sensor involved was unknown. Using in vitro models we find aberrant activation of the STING pathway due to deficient retrograde but probably not intra-Golgi transport. Further we find the upstream cytosolic DNA sensor cGAS as essentially required to drive type I IFN signalling. Genetic deletion of COPI subunits COPG1 or COPD similarly induces type I IFN activation in vitro, which suggests that inflammatory diseases associated with mutations in other COPI subunit genes may exist. Finally, we demonstrate that inflammation in COPA syndrome patient peripheral blood mononuclear cells and COPI-deficient cell lines is ameliorated by treatment with the small molecule STING inhibitor H-151, suggesting targeted inhibition of the cGAS/STING pathway as a promising therapeutic approach.

The effect of CRISPR/Cas9-mediated genetic deletion of NLRP3 (a), PKR (b), MAVS (c), UNC93B1 (d) in COPA deficient THP-1 cells was assessed by immunoblot analysis of phosphorylated STAT1 (pSTAT1) and transcription analysis of TNF, IFNB1, ISG15 and UNC93B1 by qRT-PCR. Data are presented as mean ± SEM from 4 independent experiments. Source data are provided at the end of the Supplementary Information file.

Supplementary Figure 3 | Inflammation in COPA deficient HeLa cells is STINGdependent.
CRISPR/Cas9 gene editing was performed to genetically delete STING and generate COPA deficient HeLa cells. Protein expression levels of COPA, STING and phosphorylated TBK1 (pTBK1) were assessed by immunoblot analysis of unstimulated cells. A representative experiment of n=2 is shown. Inflammatory pathway activation was also investigated by qRT-PCR analysis of proinflammatory gene transcription at baseline. Data are presented as mean from n=2 independent experiments showing individual data points. Source data are provided at the end of the Supplementary Information file.

Supplementary Figure 4 | STING-GFP co-localizes with the dispersed Golgi in COPA deficient HeLa cells.
Parental and COPA deficient HeLa cells stably reconstituted with STING-GFP were Doxtreated for 72 hrs, fixed and stained for COPA (a), GM130 (b) or KDEL (c). Localization of activated STING-GFP (green) in parental HeLa cells transfected with HT-DNA (2 µg/ml, 2 hrs) results in STING-GFP accumulation at the Golgi (GM130), which is shown as positive control. Representative images, n=1, scale bar 10 µm, white arrows indicate COPA deficient cells.

Supplementary Figure 5 | Deletion of COPA results in Golgi dispersal.
Immunofluorescence analysis of parental and COPA deficient HeLa cells after 72 hrs of Dox treatment stained for COPA (cyan), cis-Golgi marker GM130 (magenta) and DAPI (blue). Pictures are representatives of n=2 independent experiments. Scale bar: 20 µm.

Supplementary Figure 6 | Phosphorylated TBK1 is elevated in monocytes isolated from COPA syndrome patients.
a) Flow cytometry pseudocolor plots exemplifying the gating strategy used to analyse PBMC samples. The monocyte population was firstly identified based on cell size (forward scatter, FSC) and granularity (side scatter, SSC) and subsequently confirmed by gating for the CD14-FITC positive and CD3-APC negative subpopulation. The phosphorylated TBK1 (pTBK1-PE) signal was analysed using histograms normalized to mode to allow overlayed comparison between different samples. b) Flow cytometry analysis comparing the pTBK1-PE in the monocytic subpopulation isolated from COPA syndrome patient PBMCs (blue) and 2 healthy individuals (HCs, black, red) without further stimulation. Histogram shows the pTBK1 signal from this experiment, dotted line indicates the isotype control. c) Column graph quantifies data shown in b) as geometric mean fluorescence intensity (MFI) of the same experiment using 2 independent HCs (line at mean), n=1.

Supplementary Figure 7 | Endogenous cGAS/STING expression in cell lines used throughout this study.
Immunoblot analysis of endogenous STING and cGAS expression levels in HEK293, HEK293T, parental HeLa and THP-1 cell lines used in this study. A representative western blot of n=2 independent experiments is shown. Source data are provided at the end of the Supplementary Information file.

Supplementary Figure 8 | Genetic deletion of adapter protein SURF4 induces inflammatory signalling in a cGAS-dependent manner.
a) Immunoblot analysis of inflammatory signalling in THP-1 parental (WT) and monoclonal cGAS -/-THP-1 cells following genetic deletion of SURF4 using 2 different sgRNAs (sg1 and sg2), n=1. b) qRT-PCR analysis of SURF4 transcription levels of cell lines used in a). c) qRT-PCR analysis of ISG transcription levels in THP-1 cell lines used in a) and b). b) and c) Data are shown as mean from 2 technical replicates, n=1. Source data for a) are provided at the end of the Supplementary Information file.

Supplementary Figure 10 | Loss of COPA does not alter cellular expression levels of STING.
Western blot analysis of STING expression levels in THP-1 cells during 96 hrs Dox treatment when COPA protein levels gradually decline. A representative result of n=3 independent experiments is shown. Source data are provided at the end of the Supplementary Information file.